HCV anti-core monoclonal antibodies

ABSTRACT

Monoclonal antibodies that specifically bind to HCV core antigen. Also provided are hybridoma cell lines which secrete these antibodies, methods for making and using these antibodies, including kits that include these antibodies.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/337,453, filed Nov. 5, 2001 (now abandoned).

BACKGROUND OF THE INVENTION

An estimated 170 million people worldwide have been infected byhepatitis C virus (HCV). In the next few years, the number of U.S.deaths for HCV-caused liver disease and cancer may overtake deathscaused by Acquired Immune Deficiency Syndrome (AIDS).

The transmission of HCV seems to require blood-to-blood contact.Carrying a single strand of ribonucleic acid (RNA), HCV contains justone gene, coding for a polyprotein that is subsequently cleaved into atleast 10 functional proteins. Clearly, the ability to test the bloodsupply for HCV is of great importance. A sensitive assay that can detectinfection at an early stage. Detection of both HCV antigens andantibodies in a single assay would therefore be advantageous.

HCV core detection assays capture HCV antigen from the HCV infectedspecimens on a solid phase coated with anti-core monoclonal antibodies.The captured core protein is detected by a binding reaction with ananti-core monoclonal antibody labeled with enzyme using standardimmunoassay technology. Availability of good monoclonal antibodies tomultiple epitopes of HCV core antigen will increase the sensitivity ofHCV antigen detection assays. For one thing, multiple antibodiesincrease the efficiency of the capture of the HCV antigen from specimensonto the solid phase. Second, using multiple antibodies for detectionincreases the sensitivity of the system by providing a cumulative highersignal. Another advantage is that the use of multiple epitoperecognizing antibodies in assays enables the assays to detect specimensinfected with different genotypes of the HCV virus.

SUMMARY OF THE INVENTION

The present invention describes a library of monoclonal antibodiesdirected against various regions of the HCV core protein. Theseantibodies recognize ten (10) different epitopes in the HCV core regionand are spread throughout the core protein. The epitopes recognized bythese monoclonal antibodies are five to eight (5-8) amino acids (“aa”)long. These antibodies in individual pairs or multiple pairs are usefulin detecting HCV core protein in patients infected with HCV.Specifically, these antibodies are advantageous in detecting HCV inblood from infected humans. Finally, these antibodies along withproperly tailored recombinant proteins can be used for the simultaneousdetection of HCV core protein and ant-HCV antibodies.

We have developed a number of anti-HCV core monoclonal antibodies. Theseantibodies have unique properties in that they recognize shortsequences, in the region of HCV core that does not have major epitopesrecognized by human anti-HCV antibodies. These antibodies can be usefulreagents in detecting HCV core antigen in blood specimens fromindividuals infected with HCV. The recognition of short epitopes bythese monoclonal antibodies and the location of these sequences in thecore region of HCV make these monoclonal antibodies very useful in thedevelopment of a HCV antigen/antibody combination assay. For thecombination assay these monoclonal antibodies are used in combinationwith core antigen that has been modified to eliminate the ability ofthese antigens to bind to these monoclonal antibodies but maintain theirability to bind human anti-HCV antibodies. Therefore, the HCV coreproteins used to capture the antibodies present in infected serum willnot react with the HCV core monoclonal antibodies used in the test tosimultaneously capture the antigens present in the infected serum. Acombination assay can detect HCV infection earlier than the currentlyemployed HCV antibody assay.

DETAILED DESCRIPTION OF THE INVENTION

The effectiveness and advantages of the invention are furtherillustrated by the following examples.

EXAMPLE 1 Immunogens

Immunogens used for raising the antibodies were a full length corerecombinant protein (“FLC”)(amino acids (aa) 1-191); c22-3, HCV coreprotein expressed in yeast as an SOD fusion protein (aa 1-120); andlarge synthetic peptides, such as ODS243(aa 70-110); and keyhole limpethemacynin (KLH) conjugated 15 mer peptides containing sequences from HCVcore antigen.

EXAMPLE 2 Immunizing Protocol

The antibodies were generated using standard procedures. See forexample, Ed Harlow and David Lane, Antibodies, A Laboratory Manual,1988, Cold Spring Harbor, Chapter 6. Mice were immunized with theimmunogens described herein. The mice were bled and screened with anumber of recombinant proteins and peptides from the HCV core sequence.Pure monoclonal antibodies were prepared by protein A affinitychromatography. The fine specificity of the monoclonal antibodies wasdetermined by binding overlapping octapeptides contained in each of thecorresponding 15 mer peptide. Most antibodies were mapped to a 6-8 aminoacid region in the HCV core protein sequence.

EXAMPLE 3 Characterization of the Antibodies

The monoclonal antibodies were characterized by ELISA using overlappingsynthetic peptides approximately 15 aa long. The antibodies were furthercharacterized for their fine specificity by peptide ELISA usingoverlapping octapeptides included in the reactive 15 mer peptide. Asummary of antibodies generated is included in the following table.

TABLE 1

The table below identifies 15 novel antibodies. The antibodies werescreened at every stage of antibody development with microtitrewellplates coated with immunogen. The immunogen used to immunize the micethat produced each strain of monoclonal antibody is identified as one ofthe following: peptide ODS 243, a large peptide further defined inExample 1; “FLC” means full length core antigen, defined in Example 1;or KLH conjugated core peptide #8, a short peptide, further definedherein. The specificity of each to a numbered peptide is shown and theamino acid sequences of each numbered peptide is identified herein.Furthermore, the epitope to which the antibody specifically binds isincluded in the last column, defined by the amino acids that encode forthe epitope.

The antibodies were deposited on Oct. 24, 2001 with the InternationalDepository Authority: American Type Culture Collection, Manassas, Va.20110-2209 USA (“ATCC”). The ATCC accession numbers are listed in thefirst column of the table below.

AG-FUSION ATCC #, SPECIFI- # clone IMMUNOGEN ISOTYPE CITY AA PTA-ODS243, Peptide IgG2b HCV core  77- 3811 7B4F11 ODS 243 peptide #8  91PTA- ODS243, Peptide IgG2a HCV core  86- 3803 1E3D12 ODS 243 peptide #9100 PTA- ODS243, Peptide IgG2b HCV core  77- 3802 7C12C4 ODS 243 peptide#8  91 PTA- core#3, FLC IgG1 HCV core 106- 3813 2A11C6 peptide #11 120PTA- Core#12, FLC IgG1 HCV core  29- 3809 1B7A1 peptide #3  43 PTA-Core#13, FLC IgG1 HCV core  39- 3805 5A12G12 peptide #4  53 PTA-Core#13, FLC IgG1 HCV core 48- 3812 4H7E7 peptide #5  62 PTA- Core#13,FLC IgG1 HCV core  58- 3806 12F4A11 peptide #6  72 PTA- Core#13, FLCIgG1 HCV core  67- 3804 14D12A12 peptide #7  81 PTA- c22-8#4, KLH IgG1HCV core  77- 3807 6D8E8 Conjugated peptide #8  91 core peptide #8 PTA-Core#12, FLC IgG2b HCV core  96- 3800 4G10G6 peptide #10 110 PTA-Core#13, FLC IgG2a HCV core 106- 3801 6E7E1 peptide #11 120 PTA-Core#13, FLC IgG2b HCV core 106- 3810 11D12A6 peptide #11 120 PTA-Core#13, FLC IgG3 HCV core 106- 3808 14B7C3 peptide #11 120 PTA-Core#12, FLC IgG1 HCV core 156- 3799 4A6H3 peptide #16 170

TABLE 2 The table below shows HCV core synthetic peptides. Peptide AminoAcid HCV protein ID # Sequence AA Location 0 MSTNPKPQKKNKRNT  1-15 SEQID NO.:1 1 KNKRNTNRRPQDVKF 10-24 SEQ ID NO.:2 2 QDVKFPGGGQIVGGV 20-34SEQ ID NO.:3 3 QIVGGVYLLPRRGPR 29-43 SEQ ID NO.:4 4 RRGPRLGVRATRKTS39-53 SEQ ID NO.:5 5 ATRKTSERSQPRGRR 48-62 SEQ ID NO.:6 6PRGRRQPIPKARRPE 58-72 SEQ ID NO.:7 7 KARRPEGRTWAQPGY 67-81 SEQ ID NO.:88 AQPGYPWPLYGNEGC 77-91 SEQ ID NO.:9 9 YGNEGCGWAGWLLSP  86-100 SEQ IDNO.:10 10 WLLSPRGSRPSWGPT  96-110 SEQ ID NO.:11 11 SWGPTDPRRRSRLNG106-120 SEQ ID NO.:12 12 SRLNGKVIDTLTCGF 116-130 SEQ ID NO.:13 13LTCGFADLMGYIPLV 126-140 SEQ ID NO.:14 14 YIPLVGAPLGGAARA 136-150 SEQ IDNO.:15 15 GAARALAHGVRVLED 146-160 SEQ ID NO.:16 16 RVLEDGVNYATGNLP156-170 SEQ ID NO.:17 17 TGNLPGCSFSIFLLA 166-180 SEQ ID NO.:18 18IFLLALLSCLTVPAS 176-190 SEQ ID NO.:19

TABLE 3 The table below shows the screening of the antibody identifiedas PTA-3811 with ELISA plates coated with HCV core peptides. PeptideOptical Density ID # in ELISA 0 0 1 0.04 2 0 3 0.01 4 0.01 5 0 6 0 70.01 8 2.5 9 0 10 0.01 11 0.01 12 0 13 0.01 14 0.01 15 0 16 0.01 17 0.0118 0.01

TABLE 4 Epitope mapping of monoclonal antibody PTA-3811 usingoverlapping octapeptides.

Sequence of AA octapeptides Location ALU (7.9) Biotin-Ahx- 75-82 0.57SEQ ID TWAQPGYP NO.:20 (7.10) Biotin-Ahx- 76-83 1.05 SEQ ID WAQPGYPWNO.:21 (8.1) Biotin-Ahx- 77-84 0.61 SEQ ID AQPGYPWP NO.:22 (8.2)Biotin-Ahx- 78-85 10.84 SEQ ID QPGYPWPL NO.:23 (8.3) Biotin-Ahx- 79-8645.89 SEQ ID PGYPWPLY NO.:24 (8.4) Biotin-Ahx- 80-87 43.03 SEQ IDGYPWPLYG NO.:25 (8.5) Biotin-Ahx- 81-88 33.81 SEQ ID YPWPLYGN NO.:26(8.6) Biotin-Ahx- 82-89 3.11 SEQ ID PWPLYGNE NO.:27 (8.7) Biotin-Ahx-83-90 0.27 SEQ ID WPLYGNEG NO.:28 (8.8) Biotin-Ahx- 84-91 0.3 SEQ IDPLYGNEGC NO.:29 (8.9) Biotin-Ahx- 85-92 0.42 SEQ ID LYGNEGCG NO.:30(9.1) Biotin-Ahx- 86-93 0.49 SEQ ID YGNEGCGW NO.:31 (9.2) Biotin-Ahx-87-94 0.6 SEQ ID GNEGCGWA NO.:32

Monoclonal antibody 7B4F11 reacted with Octapeptides 8.2, 8.3, 8.4, 8.5in a chemiluminescence ELISA measured in arbitrary light units (ALU).Based on the reactivity of the octapeptides the monoclonal epitope iscentered around the sequence PWPL (SEQ ID NO.: 33).

The above examples are meant to illustrate, but not to limit, the scopeand spirit of the invention.

It is to be understood that numerous changes and modifications may bemade therein without departing from the scope and intent of theinvention.

For example, it would be understood by one skilled in the art that allof the monoclonal antibodies disclosed herein can be used individuallyor as combinations as capture regents or as detecting reagents for thedetection of HCV core antigen in a HCV core antigen assay or an HCVantibody/antigen combination assay.

1. A hybridoma cell line corresponding to the ATCC number selected fromthe group consisting of PTA-3799, PTA-3800, PTA-3801, PTA-3802,PTA-3803, PTA-3804, PTA-3805, PTA-3806, PTA-3807, PTA-3808, PTA-3809,PTA-3810, PTA-3811, PTA-3812, and PTA-3813.
 2. A monoclonal antibodysecreted by any one of the hybridoma cell lines claimed in claim
 1. 3.Assay kits that contain one or more of the monoclonal antibodies claimedin claim
 2. 4. A method for the detection of HCV core antigenscomprising: 1) contacting a sample that may contain said HCV coreantigens with one or more of the monoclonal antibodies claimed in claim2; and 2) detecting the presence of immune complexes as an indication ofthe presence of HCV core antigen.